5.5 Drinking Water Microbiology & the Revised Total Coliform Rule
Key Takeaways
- Total coliforms are an indicator of treatment or distribution integrity rather than a direct health hazard, while the presence of E. coli indicates fecal contamination and is a health-based violation.
- Under the Revised Total Coliform Rule there is no total coliform maximum contaminant level; instead a positive routine sample triggers repeat sampling and, if criteria are met, a Level 1 or Level 2 assessment.
- An E. coli maximum contaminant level violation occurs when a routine sample is E. coli positive and a repeat is total coliform positive, or a routine sample is total coliform positive and a repeat is E. coli positive, and it requires Tier 1 public notification within 24 hours.
- A total coliform positive sample requires three repeat samples within 24 hours, taken at the original site, within five service connections upstream, and within five service connections downstream.
- Cryptosporidium oocysts are highly resistant to free chlorine, which is why physical removal by filtration and inactivation by ultraviolet light rather than chlorination are the recognized barriers.
Drinking Water Microbiology & the Revised Total Coliform Rule
The ABC Water Treatment Class IV outline puts 11 of 17 Laboratory Analysis items at the recall level - meaning this content is memorization territory, and microbiology is a large share of it. It also lists "determine if water quality violations have occurred" and "notify the public according to regulatory requirements" in the administrative area, both of which route through the Revised Total Coliform Rule.
1. Indicator Organism Theory
Testing every drinking water sample for every pathogen is impossible - pathogens are episodic, present in small numbers, and expensive to detect. Instead, the regulations rely on indicator organisms.
| Organism group | What it indicates |
|---|---|
| Total coliforms | A broad group of gram-negative, non-spore-forming rods that ferment lactose. Found in soil, vegetation, and water as well as in feces. Not a health indicator; an indicator of treatment or distribution system integrity. |
| Fecal coliforms | The thermotolerant subset that grows at 44.5 degrees C. More specific to fecal sources; still used heavily in wastewater. |
| Escherichia coli | Almost exclusively of fecal origin. The health-based indicator in drinking water. |
| Heterotrophic plate count (HPC) | Total culturable bacteria; not a pathogen indicator but a measure of general biological activity and residual effectiveness. |
The ideal indicator is present whenever pathogens are present, absent when they are not, more numerous and more persistent than the pathogens, harmless, and cheap to detect. Total coliforms meet most of those criteria; the fact that they also come from soil and vegetation is exactly why a total coliform positive result triggers an investigation rather than an automatic health violation.
2. Analytical Methods
| Method | Procedure | Result format |
|---|---|---|
| Membrane filtration (MF) | 100 mL filtered through a 0.45 micron membrane; membrane placed on selective medium (m-Endo for total coliform, m-FC for fecal); incubated 24 h at 35 degrees C (44.5 degrees C for fecal) | Colonies per 100 mL; total coliforms show a golden-green metallic sheen on m-Endo; fecal coliforms show blue colonies on m-FC |
| Enzyme substrate / defined substrate (Colilert) | Reagent added to a 100 mL sample, incubated 24 h at 35 degrees C | Yellow = total coliform (beta-galactosidase cleaving ONPG); blue fluorescence under 365 nm UV = E. coli (beta-glucuronidase cleaving MUG). Quanti-Tray format gives an MPN |
| Multiple tube fermentation (MTF) | Presumptive, confirmed, and completed phases in lauryl tryptose and brilliant green bile broths with Durham tubes | Most probable number (MPN) per 100 mL; gas production is the presumptive positive |
| Presence/absence (P/A) | Single bottle, single result | Present or absent - which is all the RTCR requires for routine compliance |
Sample collection requirements
- Sterile 100 mL bottle containing sodium thiosulfate to neutralize the chlorine residual at the moment of collection; without it, chlorine keeps killing organisms in transit and produces a false negative.
- Holding time: 30 hours from collection to analysis for compliance samples (some states allow up to 48 hours with justification), transported at below 10 degrees C.
- Flush the tap, remove aerators and hoses, flame or disinfect the tap if practical, and do not sample from a leaking faucet or a swing spout.
- Record the disinfectant residual at the same time and place. A coliform positive with a good residual is a very different investigation than one with no residual.
3. The Revised Total Coliform Rule (RTCR)
The RTCR replaced the 1989 Total Coliform Rule in April 2016. The structural change matters:
There is no maximum contaminant level for total coliforms under the RTCR. A total coliform positive result is a trigger, not a violation. The only microbial MCL is for E. coli.
Monitoring
- Every system must have a written sample siting plan identifying routine and repeat sample locations, subject to state review.
- Community water systems monitor at a frequency based on population served - from 1 sample per month for the smallest systems up to hundreds per month for large ones.
- Groundwater systems serving 1,000 or fewer people may qualify for quarterly monitoring, with the state able to increase frequency after a positive.
Repeat sampling
A total coliform positive routine sample requires three repeat samples within 24 hours:
- At the original sampling location;
- Within five service connections upstream; and
- Within five service connections downstream.
(Systems collecting one routine sample per month or fewer collect four repeats.)
The E. coli MCL violation
An E. coli MCL violation occurs when:
- A routine sample is E. coli positive and any repeat sample is total coliform positive; or
- A routine sample is total coliform positive and any repeat sample is E. coli positive; or
- The system fails to take all required repeat samples after an E. coli positive routine sample; or
- The system fails to analyze for E. coli when any repeat sample is total coliform positive.
An E. coli MCL violation requires Tier 1 public notification within 24 hours.
Assessments - the heart of the rule
The RTCR is a "find and fix" rule. Rather than penalizing a positive result, it requires the system to look for the cause.
| Assessment | Triggered by | Who conducts it | Deadline |
|---|---|---|---|
| Level 1 | More than 5.0 percent of routine and repeat samples in a month are total coliform positive (for systems taking 40 or more samples per month); or two or more total coliform positive samples in a month (for systems taking fewer than 40); or failure to take every required repeat sample | The system itself | Submit within 30 days of learning the trigger was exceeded |
| Level 2 | An E. coli MCL violation; or a second Level 1 trigger within a rolling 12-month period | The state or a state-approved party | Submit within 30 days |
Both assessments must evaluate at minimum: atypical monitoring events, changes in distribution system maintenance and operation including water storage, source and treatment considerations, existing water quality data, and inadequacies in sampling practice. Sanitary defects found must be corrected, and the correction reported - within 30 days if already completed, or with a state-approved schedule if not.
Failure to conduct a required assessment, or to correct identified sanitary defects, is a treatment technique violation.
Seasonal systems
Systems that are not year-round (campgrounds, seasonal restaurants) must complete a state-approved start-up procedure before serving water each season and certify its completion.
4. Heterotrophic Plate Count
HPC measures total culturable heterotrophic bacteria, reported as colony forming units per mL, usually by pour plate, spread plate, or membrane filtration on R2A agar.
- It is not a pathogen indicator. No health-based limit exists.
- Under the Surface Water Treatment Rule, an HPC below 500 CFU/mL may be used in place of a detectable disinfectant residual for the purpose of demonstrating that the distribution system residual requirement is met.
- Its real value is as a trend. Rising HPC in a zone signals loss of residual, biofilm activity, nitrification, or excessive water age - and it does so before coliforms appear.
5. Pathogens Operators Must Know
| Pathogen | Type | Key operational fact |
|---|---|---|
| Cryptosporidium parvum / hominis | Protozoan (oocyst, 4 to 6 micron) | Extremely chlorine resistant - practical inactivation requires UV or ozone; the primary barrier is physical removal by filtration. Drives LT2ESWTR |
| Giardia lamblia | Protozoan (cyst, 8 to 14 micron) | Chlorine resistant but far less so than Crypto; 3-log removal/inactivation required |
| Legionella pneumophila | Bacterium | Grows in warm water and biofilm, especially in premise plumbing and cooling towers; controlled by residual, temperature management, and reducing water age |
| E. coli O157:H7 | Bacterium | Pathogenic strain; the reason E. coli presence is treated as acute |
| Enteric viruses (hepatitis A, norovirus, rotavirus, enterovirus) | Virus | 4-log removal/inactivation required; very small, so filtration alone is insufficient - disinfection carries the load |
| Naegleria fowleri | Amoeba | Rare but fatal; associated with warm water and low residual; nasal exposure route |
| Cyanotoxins (microcystin, cylindrospermopsin) | Algal toxin | Not an organism but a bloom product; PAC, GAC, and ozone are the treatment options; do not use copper algaecide during an active toxic bloom because cell lysis releases the toxin |
Log removal arithmetic, briefly: 1-log = 90 percent, 2-log = 99 percent, 3-log = 99.9 percent, 4-log = 99.99 percent. A conventional filtration plant is normally credited 2.5-log Giardia and 2-log virus removal for the treatment train, with the disinfection CT making up the remainder to reach 3-log and 4-log respectively.
A routine distribution sample is total coliform positive. Under the Revised Total Coliform Rule, what must the system do and where must the samples be taken?
A water system incurs its second Level 1 assessment trigger within a rolling 12-month period. What is required?
Why does the Long Term 2 Enhanced Surface Water Treatment Rule emphasize physical removal and ultraviolet light rather than chlorination as the barrier against Cryptosporidium?